In this paper we have engineered the targeting of ScFv fragments to mitochondria and demonstrated that this can occur efficiently. This extends the range of subcellular compartments where antibody domains can be targeted in order to interfere with the action of the corresponding antigen. Moreover, we have compared the redox state of ScFv fragments targeted to the secretory compartment, the cytosol and the mitochondria, and demonstrated that cysteine residues in ScFv targeted to the secretory compartments and to the mitochondria are oxidized. On the contrary, cytosolic antibody domains are expressed in a reduced state, which is probably the reason for their lower expression levels. These pitfalls, however, do not prevent their successful utilization for intracellular immunization.
In vertebrates, the mRNAs encoding ribosomal proteins, as well as other proteins implicated in translation, are characterized by a 5'-untranslated region (5'-UTR), including a stretch of pyrimidines at the 5'-end. The 5'-terminal oligopyrimidine (5'-TOP) sequence, which is involved in the growth-dependent translational regulation characteristic of this class of genes (so-called TOP genes), has been shown to specifically bind the La protein in vitro, suggesting that La might be implicated in translational regulation in vivo. In order to substantiate this hypothesis, we have examined the effect of La on TOP mRNA translational control in both stable and transient transfection experiments. In particular we have constructed and analyzed three stably transfected Xenopus cell lines inducible for overexpression of wild-type La or of putative dominant negative mutated forms. Moreover, La-expressing plasmids have been transiently co-transfected together with a plasmid expressing a reporter TOP mRNA in a human cell line. Our results suggest that in vivo La protein plays a positive role in the translation of TOP mRNA. They also suggest that the function of La is to counteract translational repression exerted by a negative factor, possibly cellular nucleic acid binding protein (CNBP), which has been previously shown to bind the 5'-UTR downstream from the 5'-TOP sequence.
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